Streptococcus pneumoniae, a gram-positive and naturally transformable organism, causes various infections in human and animal such as bacterial pneumonia, otitis media and meningitis (Willett, H. P. 1992. Streptococcus pneumoniae. In Zinsser Microbiology. Joklik, W. K., Willet, H. P., Amos, D. B. and Wilfert, C. M., (eds). Prentice-Hall International, London, pp. 432-442). It is known that appearance of multi-drug resistant bacteria makes it difficult to treat infections caused by Streptococcus pneumoniae using antibiotics. 23-valent polysaccharide vaccines (Pneumovax 23 (Merck) and Pnu-Imune 23 (Wyeth-Lederle)), which comprise capsular polysaccharides (CPS) as an effective antigen, are commercially available to prevent pneumococcal infections. However, these vaccines have disadvantages in that they were not effective due to low antibody production rate when given to infants and young children, and in that they have no memory response. 7-valent conjugate vaccine (Prevnar (Wyeth-Lederle)), which is made by conjugating 7 types of CPS to a carrier protein, has been developed to solve the disadvantages of 23-valent vaccines as mentioned above. However, this vaccine is very restrictly applied as a vaccine to prevent pneumoccocal infections, since it is very expensive and has protective effect against only 7 types among 95 types or more of the pneumococcus. Therefore, there have been attempts to develop a vaccine with a protein having high antigenicity to prevent pneumococcal infections. Pneumolysin (Ply) toxoid is known as a virulence factor of the pneumococcus that binds to cholesterol of host cell to form pore in the cell, and thus there have been attempts to develop vaccines using an attenuated pneumolysin (PdB). However, pneumolysin has a very high in vivo and in vitro toxicity. Also, the PdB was not effective when given alone, and elicited increased survival rate against the pneumoccocal infections only when given in combination with other virulence factors such as Pneumococcal Surface Protein A (PspA), Choline Binding Protein (CbpA), Pneumococcal Surface Adhesin A (PsaA), LytA (Ogunniyi, A. D. et al., 2000, Immunization of mice with combinations of pneumococcal virulence proteins elicits enhanced protection against challenge with Streptococcus pneumoniae. Infect. Immun. 68:3028-3033). Thus, since candidate antigen proteins in conventional vaccines for prevention of pneumoccocal infections have low antigenicity or have no protective effects against all serotypes of the pneumococcus, there remains a need to develop attenuated vaccines as well as candidate antigen proteins which have high immunogenicity and are conservatively present in all types of the pneumococcus.
The pneumococcus is carried in the nasopharynx of healthy individuals, and this is a major reservoir for pneumococcal infections. Pneumococci are subject to a number of environmental stresses in vivo. Change of environmental niche in the host, such as penetration of pneumococci from the nasopharynx into the bloodstream, can trigger dramatic changes in morphology as well as gene expression. For example, pneumococci in the nasopharynx have been shown to be predominantly of a transparent colony phenotype and tend to express less capsule and more choline binding protein A (CbpA), whereas pneumococci in the bloodstream are predominantly of the opaque colony morphology and tend to produce more capsule and less CbpA (Kim, J. O. et al., 1998. Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae. J. Infect. Dis. 177:368-377). Furthermore, S. pneumoniae encounters heat stress during its pathogenic course after penetration from the nasal mucosa (30 to 34° C.) (Lindemann, J. et al., 2002, Nasal mucosal temperature during respiration. Clin. Otolaryngol. 27:135-139) into blood and/or meninges (37° C.). This temperature shift may serve as a key trigger for a rapid, transient increase in synthesis of a highly conserved set of proteins referred to as heat-shock proteins (HSPs) (Neidhardt, F. C. et al., and R. A. VanBogelen. 1987. Heat shock response, p. 1334-11345. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), E. coli and Salmonella typhimurium: Cellular and molecular biology. ASM Press, Washington, D.C). HSPs protect bacteria against such adverse effects as elevated temperatures, exposure to ethanol, oxidative stresses, or heavy metals thus increasing their survival rate. Therefore, a thorough understanding of the heat shock response could provide useful information on adaptation of the pneumococcus to the hostile environment it encounters.
HSPs can be classified into Hsp100, Hsp70, Hsp60, and small Hsp families depending on molecular weight, and are ubiquitously present in prokaryotes and eukaryotes. One of the HSPs, hsp100/Clp (caseinolytic protease) family, is present as a 104-kDa protein in eukaryotes, but as an 80-95-kDa protein in prokaryotes. It carries out a chaperone function and is also involved in proteolysis thereby removing damaged and denatured proteins. Proteolysis by Clp requires a serine-type peptidase ClpP subunit and a regulatory ATPase subunit (Schirmer, E. C., et al., 1996. HSP100/Clp proteins: a common mechanism explains diverse functions. Trends Biochem. Sci. 21:289-296). Regulatory Clp subunit proteins can be assigned, in general, to two classes: class I, which comprises clpA, B, C, and D, contains two ATP-binding regions; class II, which comprises clpM, N, X, and Y, contains only one ATP-binding region. Clps have been classified by the size of the central spacer segment, the need for gaps in aligning the overall sequences, and sequence similarities in the well-conserved regions, and in the N- and C-terminal segments, the variable leader regions have very different sequences in each subfamily (Supra, Schirmer, E. C., et al., 1996).
Although substantial progress has been made on understanding the mechanisms of action of the Clp family in Gram-negative bacteria such as E. coli, little is known about Clp in Gram-positive bacteria. The clpP gene and clpC operon are negatively regulated by CtsR, which recognizes a directly repeated operator sequence (A/GGT CAA ANA NA/GG TCA AA), but clpX does not have this sequence and their specific mechanisms of action have not been determined in detail (Derre, I., et al., 2000. The CtsR regulator of stress response is active as a dimer and specifically degraded in vivo at 37° C. Mol. Microbiol. 38:335-347).
Since a variety of environmental signals including temperature and nutrient availability can control the expression of virulence factors, we previously examined the protein profiles of the heat shock response in pneumococci after exposure of the cells to several stresses. The major proteins induced by heat shock were 62-, 72-, and 84-kDa in size, identified subsequently as GroEL, DnaK, and ClpL, respectively. However, pulse-labeling of proteins with [35S]-methionine revealed that certain conditions which are known to induce stress responses in E. coli and B. subtilis failed to induce any high molecular weight HSPs such as GroEL and DnaK homologues. However, a temperature shift from 30 to 37° C. in vitro, similar to that encountered by S. pneumoniae after translocation from the nasal mucosa to the lungs, triggered induction of DnaK and GroEL (Choi, I. H., et al., 1999. Limited stress response in Streptococcus pneumoniae. Microbiol. Immunol. 43: 807-812). The nucleotide sequences of ClpL from several Gram-positive organisms are known (L. lactis [X62333]; S. aureus [AP003365, AP003137]; S. pyogenes [AE006538, AE004092]; Lactobacillus rhamnosus [AF323526]), but functional studies on ClpL have been limited. Recently in S. pneumoniae, the clpP− mutant was sensitive to high temperature, H2O2 and puromycin, and attenuated virulence significantly (Robertson, G. T., et al., 2002. Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence. J. Bacteriol. 184:3508-3520). Specific roles of other heat shock genes, clpC, clpE, and clpX have not been fully elucidated (Charpentier, E. et al., 2000. Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC. Mol Microbiol 37:717-726; Chastanet, A., et al., 2001. Regulation of Streptococcus pneumoniae clp genes and their role in competence development and stress survival. J. Bacteriol. 183:7295-7307).
Accordingly, in order to develop antigen proteins which are present universely in all types of the pneumococcus, and vaccines using the same, we investigated the effect of heat shock on ClpL and ClpP synthesis and evaluated the impact of clpL− and clpP− mutation on in vitro expression of key pneumococcal virulence genes. Furthermore, the effect of clpL− and clpP− mutation on the virulence of S. pneumoniae was evaluated in a mouse intraperitoneal challenge model. Here we demonstrate that the heat shock process induced expression of pneumolysin (Ply) and modulated the expression of other virulence factors in wild-type pneumococci. We also show that clpP− mutation resulted in an increase in mRNA expression, but not in the activity of Ply at elevated temperatures. Subsequently, we investigated further the underlying mechanism by which ClpP attenuates virulence and determined whether ClpP immunization could protect the mice against the challenge with virulent S. peumoniae, thereby we completed this invention.